STRUCTURAL ANALYSIS OF RIBOSOME BINDING ELEMENTS OF THE 3´ UTR IN TWO POSITIVE-STRAND PLANT RNA VIRUSES

Turnip crinkle virus (TCV) and Pea enation mosaic virus (PEMV) are two positive (+)-strand RNA viruses that are used to investigate the regulation of translation and replication due to their small size and simple genomes. Both viruses contain cap-independent translation elements (CITEs) within their 3´ untranslated regions (UTRs) that fold into tRNA-shaped structures (TSS) according to nuclear magnetic resonance and small angle x-ray scattering analysis (TCV) and computational prediction (PEMV). Specifically, the TCV TSS can directly associate with ribosomes and participates in RNA-dependent RNA polymerase (RdRp) binding. The PEMV kissing-loop TSS (kl-TSS) can simultaneously bind to ribosomes and associate with the 5´ UTR of the viral genome. Mutational analysis and chemical structure probing methods provide great insight into the function and secondary structure of the two 3´ CITEs. However, lack of 3-D structural information has limited our understanding of their functional dynamics. Here, I report the folding dynamics for the TCV TSS using optical tweezers (OT), a single molecule technique. My study of the unfolding/folding pathways for the TCV TSS has provided an unexpected unfolding pathway, confirmed the presence of Ψ3 and hairpin elements, and suggested an interconnection between the hairpins and pseudoknots. In addition, this study has demonstrated the importance of the adjacent upstream adenylate-rich sequence for the formation of H4a/Ψ3 along with the contribution of magnesium to the stability of the TCV TSS. In my second project, I report on the structural analysis of the PEMV kl-TSS using NMR and SAXS. This study has re-confirmed the base-pair pattern for the PEMV kl-TSS and the proposed interaction of the PEMV kl-TSS with its interacting partner, hairpin 5H2. The molecular envelope of the kl-TSS built from SAXS analysis suggests the kl-TSS has two functional conformations, one of which has a different shape from the previously predicted tRNA-shaped form. Along with applying biophysical methods to study the structural folding dynamics of RNAs, I have also developed a technique that improves the production of large quantities of recombinant RNAs in vivo for NMR study. In this project, I report using the wild-type and mutant E.coli strains to produce cost-effective, site-specific labeled, recombinant RNAs. This technique was validated with four representative RNAs of different sizes and complexity to produce milligram amounts of RNAs. The benefit of using site-specific labeled RNAs made from E.coli was demonstrated with several NMR techniques.

Using Habitat Equivalency Analysis to Assess the Cost Effectiveness of Restoration Outcomes in Four Institutional Contexts

At the national level, with a fixed amount of resources available for public investment in the restoration of biodiversity, it is difficult to prioritize alternative restoration projects. One way to do this is to assess the level of ecosystem services delivered by these projects and to compare them with their costs. The challenge is to derive a common unit of measurement for ecosystem services in order to compare projects which are carried out in different institutional contexts having different goals (application of environmental laws, management of natural reserves, etc.). This paper assesses the use of habitat equivalency analysis (HEA) as a tool to evaluate ecosystem services provided by restoration projects developed in different institutional contexts. This tool was initially developed to quantify the level of ecosystem services required to compensate for non-market impacts coming from accidental pollution in the US. In this paper, HEA is used to assess the cost effectiveness of several restoration projects in relation to different environmental policies, using case studies based in France. Four case studies were used: the creation of a market for wetlands, public acceptance of a port development project, the rehabilitation of marshes to mitigate nitrate loading to the sea, and the restoration of streams in a protected area. Our main conclusion is that HEA can provide a simple tool to clarify the objectives of restoration projects, to compare the cost and effectiveness of these projects, and to carry out trade-offs, without requiring significant amounts of human or technical resources.

Crowdfunding for entrepreneurship: an exploratory multivariate analysis concerning kickstarter and indiegogo platforms

The general purpose of this work is to describe and analyse the financing phenomenon of crowdfunding and to investigate the relations among crowdfunders, project creators and crowdfunding websites. More specifically, it also intends to describe the profile differences between major crowdfunding platforms, such as Kickstarter and Indiegogo. The findings are supported by literature, gathered from different scientific research papers. In the empirical part, data about Kickstarter and Indiegogo was collected from their websites and also complemented with further data from other statistical websites. For finding out specific information, such as satisfaction of entrepreneurs from both platforms, a satisfaction survey was applied among 200 entrepreneurs from different countries. To identify the profile of users of the Kickstarter and of the Indiegogo platforms, a multivariate analysis was performed, using a Hierarchical Clusters Analysis for each platform under study. Descriptive analysis was used for exploring information about popularity of platforms, average cost and the most popular area of projects, profile of users and future opportunities of platforms. To assess differences between groups, association between variables, and answering to the research hypothesis, an inferential analysis it was applied. The results showed that the Kickstarter and Indiegogo are one of the most popular crowdfunding platforms. Both of them have thousands of users and they are generally satisfied. Each of them uses individual approach for crowdfunders. Despite this, they both could benefit from further improving their services. Furthermore, according the results it was possible to observe that there is a direct and positive relationship between the money needed for the projects and the money collected from the investors for the projects, per platform.

Low cost 3D global instability analysis and flow sensitivity based on dynamic mode decomposition and high-order numerical tools

We explore the recently developed snapshot-based dynamic mode decomposition (DMD) technique, a matrix-free Arnoldi type method, to predict 3D linear global flow instabilities. We apply the DMD technique to flows confined in an L-shaped cavity and compare the resulting modes to their counterparts issued from classic, matrix forming, linear instability analysis (i.e. BiGlobal approach) and direct numerical simulations. Results show that the DMD technique, which uses snapshots generated by a 3D non-linear incompressible discontinuous Galerkin Navier?Stokes solver, provides very similar results to classical linear instability analysis techniques. In addition, we compare DMD results issued from non-linear and linearised Navier?Stokes solvers, showing that linearisation is not necessary (i.e. base flow not required) to obtain linear modes, as long as the analysis is restricted to the exponential growth regime, that is, flow regime governed by the linearised Navier?Stokes equations, and showing the potential of this type of analysis based on snapshots to general purpose CFD codes, without need of modifications. Finally, this work shows that the DMD technique can provide three-dimensional direct and adjoint modes through snapshots provided by the linearised and adjoint linearised Navier?Stokes equations advanced in time. Subsequently, these modes are used to provide structural sensitivity maps and sensitivity to base flow modification information for 3D flows and complex geometries, at an affordable computational cost. The information provided by the sensitivity study is used to modify the L-shaped geometry and control the most unstable 3D mode.

Age-Dependent Fragility and Life-Cycle Cost Analysis of Timber and Steel Distribution Poles Subjected to Hurricanes

Power distribution systems are susceptible to extreme damage from natural hazards especially hurricanes. Hurricane winds can knock down distribution poles thereby causing damage to the system and power outages which can result in millions of dollars in lost revenue and restoration costs. Timber has been the dominant material used to support overhead lines in distribution systems. Recently however, utility companies have been searching for a cost-effective alternative to timber poles due to environmental concerns, durability, high cost of maintenance and need for improved aesthetics. Steel has emerged as a viable alternative to timber due to its advantages such as relatively lower maintenance cost, light weight, consistent performance, and invulnerability to wood-pecker attacks. Both timber and steel poles are prone to deterioration over time due to decay in the timber and corrosion of the steel. This research proposes a framework for conducting fragility analysis of timber and steel poles subjected to hurricane winds considering deterioration of the poles over time. Monte Carlo simulation was used to develop the fragility curves considering uncertainties in strength, geometry and wind loads. A framework for life-cycle cost analysis is also proposed to compare the steel and timber poles. The results show that steel poles can have superior reliability and lower life-cycle cost compared to timber poles, which makes them suitable substitutes.